BrdU Antibody Staining Protocol for Immunohistochemistry


Description: To measure DNA synthesis or cell proliferation, 5-bromo2’-deoxy-uridine (BrdU) can be incorporated into DNA in place of thymindine. Cells, which have incorporated BrdU into DNA, can be quickly detected using a monoclonal antibody against BrdU. The binding of the antibody is achieved by denaturation of the DNA. This is usually obtained by exposing the cells to acid, or heat.


Primary Antibody

Name: BrdU Antibody

Clone: BU1/75, ICR1, Rat anti-BrdU

Supplier: Accurate Chemical & Scientific

Catalog Number: OBT0030

Dilution: 1:100 using IHC-TekTM Antibody Diluent (Cat# IW-1000 or IW-1001) to reduce background and unspecific staining and serum blocking step is NOT needed.

Incubation Time/Temp: 60min/room temperature

Antigen Retrieval
Device: IHC-TekTM Epitope Retrieval Steamer Set (Cat# IW-1102)
Buffer/pH value: IHC-TekTM Epitope Retrieval Solution (Cat# IW-1100) or 2N HCl (see Note below for details)
Heat/Cool Temperature: 95-100 ºC/room temperature
Heat/Cool Time: 20 minutes/20 minutes

Detection Methods
Standard Method: ABC Method or LSAB Method
Enhanced Method: Polymeric Methods

Chromogen Substrate
Reagent: DAB
Incubation Time/Temperature: 1-3 minutes/room temperature

Reagent: Mayer's Hematoxylin
Staining Time: 30 seconds

Staining Pattern: Nuclear
Images: Search image

Additional Information:
Tissue Type: BrdU incorporated tissues
Fixation: Formalin-fixed paraffin sections
Positive Control: BrdU incorporated tissues
Negative Control: Omit primary antibody, isotype control, absorption control
Blocking: 2-5% normal serum to reduce unspecific background staining; 0.5-3% H2O2 to block endogenous peroxidase activity; avidin/biotin to block endogenous biotin activity if necessary


Denature DNA by incubating sections in 2N HCl for 30 minutes at 37 °C, and neutralize the acid by immersing sections in 0.1M borate buffer for 2x5 min.


2N HCl:

10N HCl --------------------------- 20 ml

Distilled water -------------------- 80 ml

Mix well.


0.1M Borate Buffer, pH 8.5:

Sodium borate (MW 381.4) ----- 3.8 g

Distilled water -------------------- 100 ml

Mix to dissolve and adjust pH to 8.5